The present invention relates to a chicken anaemia virus (CAV), a vaccine comprising a CAV and a method for the preparation of a CAV vaccine.
Chicken anaemia virus (CAV) is the causative agent of a disease known as avian infectious anaemia, anaemia dermatitis syndrome or blue-wing disease and was first described by Yuasa et al. in 1979 (Avian Diseases 23, 366-385, 1979).
Most outbreaks of naturally occurring CAV-induced disease have been reported in young chickens. The disease is acute and the first signs usually occur at 10-14 days of age. This clinical disease is characterised by a sudden increase in mortality, usually around 5-10%, but up to 60% has been reported. Peak mortality occurs within 5 to 6 days of onset of disease. Further clinical signs include depression and anorexia. Moreover, severe anaemia, haemorrhages throughout the body, atrophy of the thymus and bursa of Fabricius and yellowish bone marrow is seen in affected chickens (McNulty, Avian Pathol. 20, 187-203, 1991).
CAV spread both horizontally and vertically in chickens. When in-lay breeders with no previous exposure to the virus become infected, CAV is transmitted vertically to the progeny. No clinical signs are seen in the breeders and there is no apparent effect on egg production, hatchability or fertility. Vertically infected progeny chicks appear normal at hatching but showed increased mortality and develop typical disease (anaemia dermatitis syndrome) from 10 to 14 days of age. Horizontal spread occurs through contact with vertically infected chickens, contaminated fomites, houses, etc. Horizontal spread to young susceptible chickens that means without maternally derived antibodies to CAV may also lead to clinical disease two weeks later.
Ensuring that parent flocks develop antibodies to CAV before onset of lay can control the anaemia dermatitis syndrome. A high level of antibodies against CAV before the onset of lay will prevent vertical transmission during lay and will provide the off spring with maternally derived antibodies, which are protective during the first few weeks after hatching against horizontal infection.
Therefore, it is important that all birds are vaccinated with a CAV vaccine before the onset of lay.
Maternal antibody to CAV has usually disappeared by about 3 weeks of age. By that time horizontal infections can take place. These infections are normally sub-clinical, however this sub-clinical infection is associated with significant economic losses due to reduced growth of the broilers (McNulty et al., Avian Diseases 35, 263-268, 1991). The sub-clinical disease may be prevented by vaccinating the chickens immediately after hatch, preferably at one-day of age.
Clearly, a need exists for a safe vaccine that induces an effective protection against the clinical and sub-clinical disease associated with CAV infections. Because the possibility of spreading of vaccine viruses to susceptible flocks of young chickens exist in practise, live CAV vaccines for parent stock vaccination should be based on CAVs of low pathogenicity. Furthermore, as young chickens are very susceptible to CAV infection, live vaccines for direct administration to young chickens requires the availability of CAV isolates of low pathogenicity which do not adversely affect the young chicks.
However, all naturally occurring CAVs isolated so far are pathogenic for young chicks (McNulty, Avian Pathology 20, 187-203, 1991; Noteborn and Koch, Avian Pathology 24, 11-31, 1995; McNulty, British Poultry Science 38, 7-13, 1997). In addition, the attenuation of CAV isolates by in vitro passages in cell culture has resulted in ambiguous results. Bulow and Fuchs (J. Vet. Med. B 33, 568-573, 1986) reported a decrease of the pathogenicity of the Cux-1 isolate after 12 passages in MDCC-MSB1 cells which was further reduced after an additional 100-112 passages. However, Yuasa (Nat. Inst. Anim. Health Quarterly 23, 13-20, 1983) and Goryo et al. (Avian Pathology 16, 149-163, 1987) found no evidence of attenuation when CAV isolates were subjected to 19 and 40 cell cultures passages, respectively. Todd et al. (Avian Pathology 24, 171-187, 1995) demonstrated attenuation of the Cux-1 isolate by passages (173 times) in MDCC-MSB1 cells, but it was established that this attenuation was not stable and reversion to virulence occurred. European patent application no. 0533294 discloses CAV isolates attenuated by passages in embryonated eggs. These isolates still display some rest-virulence for one-day-old chicks and, hence, are not particularly suited for vaccinating chicks younger than 1 week-of-age. In addition, these attenuated CAV isolates induce lesions in chicken embryos that make these vaccine viruses less suited for in ovo vaccination.
The chicken has been considered as the natural host for CAV. CAV was not found in a survey of UK turkey and duck sera and one-day-old turkey poults inoculated with CAV did not show clinical signs of anaemia and did not develop antibodies to the virus (McNulty et al., Avian Pathology 17, 315-324, 1988 and McNulty, Avian Pathology 20, 187-203, 1991). Only recently Farkas et al. (Avian Pathology 27, 316-320, 1998) reported that CAV antibodies were detected in a species (i.e. quail) other than chicken.
It is an object of the present invention to provide additional CAVs of low pathogenicity which can advantageously be used for the preparation of a live CAV vaccine, e.g. for broiler vaccination.
Another object of the present invention is to provide CAVs of low pathogenicity, in particular non-pathogenic CAVs which can be used for vaccinating birds most susceptible to CAV, e.g. for in ovo vaccination or vaccination of one-day-old birds.
It has now been found that these objects can be met by providing a chicken anaemia virus (CAV), characterised in that the virus is neutralised by a reference sample comprising monoclonal antibody R2 secreted by a hybridoma cell line, a sample of which is deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury, UK, on Feb. 3, 2000 under accession no. 00020304.
Surprisingly, it has been found that naturally occurring strains of CAV of low pathogenicity exist. The CAVs according to this invention exhibit significantly reduced pathogenicity in one-day-old chickens if compared with naturally occurring CAV strains described so far, as determined by the ability of the virus to induce thymus atrophy, pale bone marrow and/or anaemia (Table 2). Similar unexpectedly, it has been found that these CAVs are isolatable from infected turkeys in the field.
The CAVs according to the invention are antigenically distinguishable from the hitherto known CAV strains isolated from infected chickens as well as from certain other CAV strains isolated from turkeys. Monoclonal antibodies (Moabs) are useful for identifying characteristics of an infectious agent, and for determining antigenic similarities and differences among different isolates of the same or similar micro-organism. In Table 1 it is shown that the CAVs according to the present invention have a reaction pattern with a Moab which is different from that observed with known chicken strains or other turkey strains with a pathogenic character.
In particular it has been found that a CAV strain according to the invention is a virus that is neutralised by a sample comprising Moab R2, in contrast to the known CAV strains isolated from chickens that are not neutralised by this Moab.
To examine whether a CAV strain is neutralised by a sample comprising Moab R2, first the neutralising antibody titre of the Moab R2 sample against the CAV strain 319 deposited at the European Collection of Animal Cell Cultures (ECACC), Salisbury, UK, on Jan. 26, 2000 under accession no. 00012608 must be established in a virus neutralisation test, as described in Example 1 below. Depending on the antibody titre, a Moab R2 reference sample is prepared by either dilution or concentration of the Moab R2 sample so that 50 xcexcl contains an antibody titre of 16 (log2) when examined against 300-1000 TCID50 per 50 xcexcl of CAV strain 319 in a virus neutralisation test as described in Example 1.
A CAV strain is considered to belong to the present invention when it is specifically neutralised in the virus neutralisation test by the Moab R2 reference sample. This means that in the virus neutralisation test the antibody titre of the Moab R2 reference sample is at least 5 (log2) per 50 xcexcl when examined against 300-1000 TCID50 per 50 xcexcl of a CAV strain.
In particular, the present invention provides a CAV strain that is neutralised by higher dilutions of the Moab R2 reference sample. CAV strains that are neutralised by higher dilutions of the Moab R2 reference sample also exhibit a lower pathogenicity for chickens. Therefore, in a preferred embodiment of the invention a CAV strain is provided that is characterised by the fact that the antibody titre of the Moab R2 reference sample against that CAV strain is at least 10 (log2) per 50 xcexcl, more preferably at least 12 (log2) per 50 xcexcl or even at least 14 (log2) per 50 xcexcl and particularly at least 16 (log2) per 50 xcexcl when examined against 300-1000 TCID50 per 50 xcexcl of a CAV strain.
A CAV strain that is neutralised by higher dilutions of the Moab R2 reference sample is essentially non-pathogenic for young chickens and for chicken embryos. Therefore, such a CAV strain is particularly suited to be used in a live CAV vaccine for administration to chickens that are most susceptible for CAV infection, such as chicken embryos or one-day-old chickens.
A most preferred strain according to the present invention is CAV strain 319, a sample of which is deposited at the ECACC under accession no. 00012608. In view of its non-pathogenic properties this strain is particularly suited as a vaccine component for immunising young chickens or chicken embryos.
The identification of the new CAV strains according to the present invention allows the preparation of live CAV vaccines with low pathogenicity which can effectively protect poultry, in particular young chickens, against disease conditions resulting from the infection by the CAV. Therefore, in a preferred embodiment of this invention a CAV strain as defined above is provided that is in a live form.
Of course, the present invention also provides a CAV strain in inactivated from. The inactivated CAV strain can be used as a basis for an inactivated vaccine particularly suited for breeder vaccination.
A CAV according to the invention can also be isolated from turkeys in the field. Briefly, a serological survey of turkey sera collected from turkey flocks can be conducted to identify serum samples that are able to neutralise CAV in a standard virus neutralisation test. An example of such a survey is outlined in Farkas et al. (1998, supra). Subsequently, CAV can be isolated from organs of turkeys, as described in Example 1. Finally, a CAV according to the present invention can be identified by examining the reaction with the monoclonal antibody R2.
If desired, the CAVs of low pathogenicity characterised above can be adapted to embryonated eggs by passaging these CAVs in embryonated eggs such that the resulting viruses are able to grow to high titres in embryonated eggs. European patent application no. 0533294 discloses that the ability of a CAV to induce embryo lesions is associated with a growth advantage and further describes how such viruses can be obtained. Therefore, the present invention also provides CAVs of the above-mentioned type, which additionally have the property to induce lesions in chicken embryos. Such CAVs are suited for vaccination in ovo vaccination of embryos of 17 days and older or post-hatch vaccination of chickens of one-day-old or older.
The invention provides in a farther aspect a vaccine for use in the protection of poultry against disease conditions, both clinical and sub-clinical, resulting from a CAV infection, comprising a CAV according to the present invention and a pharmaceutical acceptable carrier or diluent.
The CAV according to the present invention can be incorporated into the vaccine as a live or inactivated virus. However, the low pathogenicity of the present CAVs make these viruses particularly suited for incorporation in a live CAV vaccine.
A vaccine according to the invention can be prepared by conventional methods such as for example commonly used for the commercially available CAV vaccines. The preparation of veterinary vaccine compositions is also described in xe2x80x9cHandbuch der Schutzimpfungen in der Tiermedizinxe2x80x9d (eds.: Mayr, A. et al., Verlag Paul Parey, Berlin und Hamburg, Germany, 1984) and xe2x80x9cVaccines for Veterinary Applicationsxe2x80x9d (ed.: Peters, A. R. et al., Butterworth-Heinemann Ltd, 1993).
Briefly, a suitable substrate is inoculated with a live CAV according to the invention and propagated until the virus replicated to a desired infectious titre or antigen mass content after which CAV containing material is harvested and formulated to a pharmaceutical composition with prophylactic activity.
Every substrate that is able to support the replication of the CAV defined above can be used to produce a vaccine according to the present invention. Suitable substrates include cell cultures, such as MDCC-MSB1 cells, chicken embryos and chickens for in vivo vaccine production.
For production on cell culture, the virus is usually propagated for 3-10 days after inoculation of the cells, after which the cell culture supernatant is harvested, and if desired filtered or centrifuged in order to remove cell debris.
Alternatively, the CAV according to the invention can be propagated in embryonated chicken eggs followed by harvesting the CAV material by routine methods such as described in European patent application no. 0533294.
The vaccine according to the invention containing the live CAV can be prepared and marketed in the form of a (frozen) suspension or in a lyophilised form. The vaccine additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
If desired, the live vaccines according to the invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are the same as mentioned below for the preparation of inactivated vaccines.
Although administration by injection, e.g. intramuscular, subcutaneous of the live vaccine according to the present invention is possible, the live vaccine is preferably administered by the inexpensive mass application techniques commonly used for poultry vaccination. These techniques include drinking water and spray vaccination.
Alternative methods for the administration of the live vaccine include in ovo, eye drop and beak dipping administration.
As the present invention provides CAVs which are substantially non-pathogenic when administered in ovo in the last quarter of the incubation period, a particularly advantageous route for administrating a vaccine according to the present invention is the in ovo administration.
Usually, the vaccine is injected into embryonated eggs during late stages of the embryonation, generally during the final quarter of the incubation period (day 15-21), preferably at day 18 of the incubation period. The mechanism of injection of the incubated eggs is not particularly critical provided that it does not unduly damage tissue and organs of the embryo. For example, a small hole is pierced with a needle (1-1xc2xd inch, about 22 gauge) attached to syringe in the large end of the shell and the vaccine is injected below the inner shell membrane and the chorioallantoic membrane. Subsequently, the vaccinated embryonated eggs are transferred to an incubator to hatch (U.S. Pat. Nos. 4,458,630, 5,427,791, WO 98/56413 and WO 95/35121). Preferably, the whole embryo vaccination process is carried out using automated vaccination systems, such as the commercially available Inovoject(copyright).
In another embodiment the present invention provides a vaccine against disease conditions resulting from CAV infection comprising the CAV in an inactivated form. The advantage of an inactivated vaccine is the elevated levels of protective antibodies of long duration that can be obtained. This property makes such an inactivated vaccine in particular suited for breeder vaccination. The preparation of an inactivated CAV vaccine according to the present invention can be obtained by routine methods well known to the person skilled in the art (such as described in European patent application no. 0533294).
A vaccine containing the inactivated CAV can, for example, comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
Preferably, an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F(copyright) or Marcol 52(copyright) or a vegetable oil such as vitamin E acetate, and saponins.
Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.
The vaccine according to the invention comprises an effective dosage of the CAV defined above as the active component, i.e. an amount of immunising CAV material that will induce immunity in the vaccinated birds or their progeny, against challenge by a virulent virus. Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
Typically, the live vaccine according to the invention can be administered in a dose of 102-109 TCID50 per bird, preferably in a dose ranging from 102-106 TCID50, and an inactivated vaccines may contain the antigenic equivalent of 104-1010 TCID50 per bird.
Although, the CAV vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys and quail may be successfully vaccinated with the vaccine. Chickens include broilers, reproduction stock and laying stock.
Because the clinical and sub-clinical disease conditions resulting from CAV infection, have been reported primarily in young chicks, in particular in broiler chickens, the present invention preferably provides a vaccine for use in the protection of broilers against CAV induced disease conditions.
The age of the animals receiving a live or inactivated vaccine according to the invention can be the same as that of the animals receiving the CAV vaccines presently known. Additionally, the low pathogenic character of the CAVs according to the present invention allows the administration of the CAV vaccine to young birds, i.e. less than two weeks of age, in particular to one-day-old birds or even to embryos by the in ovo route in the final quarter of the incubation period. For example, young birds, e.g. broilers, may be vaccinated directly from one-day-old onwards with the live vaccine according to the invention to prevent sub-clinical disease resulting from horizontal transmission of CAV. Vaccination of parent stock, such as broiler breeders, can be done with a live or inactivated vaccine according to the invention or with a protocol comprising a combinations of both vaccines. The advantage of these types of immunisation programmes includes the immediate protection of one-day-old progeny provided by maternally derived antibodies vertically transmitted to the young birds. A typical breeder vaccination programme includes the vaccination of the breeders from 6-weeks of age onwards with a live vaccine, or the vaccination between 14-18 weeks of age with an inactivated vaccine.
The present invention also provides a combination vaccine comprising, in addition to the CAV according to the invention, one or more vaccine components of other pathogens infectious to poultry.
Preferably, the combination vaccine comprises one or more (inactivated) vaccine strains of Mareks disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), fowl adenovirus (FAV), EDS virus, turkey rhinotracheitis virus (TRTV), infectious laryngotracheitis virus (ILTV) and reovirus.
In particular, the present invention provides a live combination vaccine comprising a CAV according to the invention and a MDV vaccine strain, such as HVT. This combination vaccine can advantageously be used for in ovo vaccination.